Measuring the presence and levels of certain molecules which are associated with cell viability is important in a number of contexts. For example, measuring levels of ATP is useful in mammalian cells for growth analysis and toxicology purposes.
Such molecules can also act as a useful marker in hygiene applications. Detection of the contaminating molecule on a surface indicates that the surface has not been cleaned or requires cleaning to the required standard.
Contamination of various products for human consumption and use by organisms such as bacteria and yeast and by cells of plant or animal origin represents an important consideration in terms of both safety and economics in a wide range of fields.
For example, water supplies, wastewater, marine environments, pharmaceutical products, cosmetic products, food, beverages, clinical samples including blood and platelet samples etc are all regularly tested for contamination by potentially harmful organisms and cells. Often, the organisms include bacterial species.
In many cases, tests are carried out based upon measuring the presence of a molecule which can be linked to the presence in the sample of a contaminant cell or organism. The most commonly detected molecule is Adenosine Triphosphate (ATP).
A routinely employed method for determining ATP levels involves the use of bioluminescence. The method uses the ATP dependency of the reaction in which light emitting luciferase catalyzes oxidation of luciferin. The method may be used to measure relatively low concentrations of ATP. Kits useful for detecting ATP using bioluminescence are commercially available from Roche, New Horizons Diagnostics Corp, Celsis etc.
A number of problems exist with respect to bioluminescence detection. For example, detection of microbial ATP only, in the presence of ATP from non-microbial sources can be a problem. This problem has been solved to a certain degree by use of filters which can separate bacteria from non-bacterial sources of ATP, thus providing a more accurate signal.
In addition, chemicals and/or metals in a sample can interfere with the bioluminescence reaction. This is of particular relevance, for example, where surface contamination is being measured following cleaning of a surface using cleaning agents. The chemical cleaning agents interfere with the luciferase catalysed reaction, and thus in some cases lead to false negative results, where microbial or other contaminant ATP is present but the bioluminescence reaction is not effective.
Ligases are well known enzymes which catalyze ligation of the 5′ end of a nucleic acid molecule to the 3′ end of a further nucleic acid molecule. The ligation reaction requires ATP in order to activate the enzyme. Ligases are commercially available and are supplied pre-loaded with an AMP molecule which allows them to catalyze ligation without activation by an ATP molecule.